One of the fundamental questions in control of gene expression in mammals is how epigenetic methylation patterns of DNA and histones are established, erased, and detected. Epigenetic regulation is a newly appreciated mechanism that profoundly influences chromatin function, which has direct relevance to a large number of human diseases from nine known imprinting-associated fertility disorders, obesity, immune response, to cancer. Mammalian DNA methylation at CpG dinucleotides is intricately connected to the methylation status of two lysine residues of histone H3: the unmodified lysine 4 (H3K4) and methylated lysine 9 (H3K9). Mammalian DNA methyltransferases, Dnmt1 and Dnmt3a, each contain a C-terminal catalytic domain and a largely uncharacterized N-terminal region including several distinct regulatory domains. Two protein lysine (K) methyltransferases (PKMTs) - G9a and Set7 - are believed to interact directly with Dnmts and modulate DNA methylation. Despite its importance, it is not known how Dnmts and PKMTs work in concert. We hypothesize that modification(s) of Dnmts themselves by PKMTs is a major component of epigenetic regulation, and may serve as a checkpoint for correct assembly of the machinery required to accurately methylate DNA. The central goal of this proposal is to determine how and where Dnmts are methylated, and how methylated Dnmts are recognized and recruited. Further, both de novo methylation of DNA CpGs by Dnmt3a, and maintenance methylation of hemimethylated DNA by Dnmt1, will be studied in parallel. I propose here four new specific aims that will determine (1) how the Set7-mediated lysine methylation marks on Dnmt1 are influenced by modifications at nearby residues, (2) how G9a-mediated methylation of Dnmt1 is recognized and recruited to the replication foci, (3) how Dnmt3a lysine methylation marks are created and recognized, and (4) the architecture of Dnmt3a-Dnmt3L heterotetramer, that is responsible for detecting both unmethylated H3K4 and CpG spacing. PUBLIC HEALTH RELEVANCE: One of the fundamental questions in control of gene expression in mammals is how epigenetic methylation patterns of DNA and histones are established, erased, and detected. Epigenetic regulation is a newly appreciated mechanism that profoundly influences chromatin function, which has direct relevance to a large number of human diseases. Serious human diseases, ranging from nine known imprinting-associated fertility disorders, obesity, immune response, to cancer, can result from defects in the methyl marking system. Alterations in this methylation can also profoundly affect infants conceived through in vitro fertilization. Thus, structural and biochemical studies directed against the emerging epigenetic regulatory enzymes may provide a method for the development of highly selective therapeutic agents that promise entirely novel approaches for the treatment of human diseases. In this proposal, we will explore questions of dynamic regulation (creating and recognizing) of DNA and histone lysine modifications and modification(s) of enzymes themselves, and biochemical crosstalk between modifications by two distinct classes of epigenetic regulators.